A fundamental control point governing growth and differentiation is the faithful duplication of the eukaryotic genome. Genetic studies in budding yeast have identified several proteins that assemble into a "pre-initiation complex" at specific chromosome locations during G1 phase of the cell cycle. Cdc6 homologues are essential components of this complex; they are necessary for DNA initiation, are modified in a cell cycle-dependent manner, and their destruction may function in the block to re-replication. I will investigate the biochemical basis of DNA replication using Xenopus egg extracts. The Xenopus egg extract supports efficient DNA replication, cell cycle progression, and nuclear envelope formation and breakdown in vitro. Using the Xenopus extract, we identified and characterized the Xenopus Cdc6 (Xcdc6) protein which, like its yeast homologues, plays a critical early step in chromosomal replication. We demonstrated that it is essential for initiation of DNA replication, coordinates the binding of other replication machinery, and its relocalization to the nuclear envelope may serve to prevent re- initiation. Using the Xenopus extract model system, I will address two major questions: 1)how does Xdc6 contribute to the initiation of DNA replication and 2)what mechanisms regulate the Xcdc6 activity? To this end, I will: 1)Map the functional domains of Xcdc6. I will address structure/function issues by characterizing various truncated and mutant forms of Xcdc6 to evaluate their activity and localization. 2)Assess the role of phosphorylation on Xcdc6 function. Xcdc6 is phosphorylated in a cell cycle-dependent manner. To investigate whether phosphorylation regulates Xcdc6 function, I will define the residues modified and evaluate the contribution of these modifications to Xcdc6 regulation and localization by characterizing appropriate point mutants. Given that Xenopus and human Cdc6 are 80 percent identical, what we learn in biochemically-tractable frog extract systems will advance our understanding of human cell-cycle control. Information derived from these studies should suggest effective strategies to combat uncontrolled cell division, which is the hallmark of cancer.